Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins
Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins
复制标题
DOI：
10.1021/pr050387a
复制
发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Liang, SP
中科院分区：
文献类型：
--
作者：
Cao, R;Li, XW;Liang, SP
为了全面鉴定大鼠肝质膜（PM）蛋白质，我们采用了一种蛋白质组学策略，即利用蔗糖密度离心结合双水相分配进行质膜分离，然后进行SDS-PAGE、质谱和生物信息学分析。Western blot分析表明，该方法可获得高纯度的质膜组分，这是成功进行质膜蛋白质组学研究的关键。用SDS-PAGE分离PM蛋白，用胰蛋白酶消化。通过nano-ESI-LC MS/MS分析，我们确定了428大鼠肝膜蛋白，其中304有一个基因本体（GO）注释指示细胞成分，后者204（67%）是已知的整合膜蛋白或膜相关蛋白。除了已知与质膜相关的蛋白质外，还鉴定了几种假设的蛋白质。这项研究不仅提供了一个工具，以研究质膜蛋白与低水平的污染，但也提供了一个数据集的蛋白质在大鼠肝脏质膜的高至中等丰度，从而允许更全面的表征膜蛋白和膜动力学更好地理解。
To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDSPAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.